Plants have long been a commercially valuable source of oil. Traditionally, plant oils were used for nutritional purposes. Recently, however, attention has focused on plant oils as sources of industrial oils, for example as replacements for, or improvements on, mineral oils. Given that oil seeds of commercially useful crops such as Brassica napus contain a variety of lipids (Hildish & Williams, “Chemical Composition of Natural Lipids”, Chapman Hall, London, 1964), it is desirable to tailor the lipid composition to better suit our needs, for example using recombinant DNA technology (Knauf, TIBtech, February 1987, 40-47).
The production of commercially desirable specific oils in plants on a large scale is limited in two ways. Some plant species make oils with very high levels of essentially pure, specific fatty acids, but these species are unable to be grown in sufficient quantities and of sufficient yield to provide a commercially valuable product. Other plant species produce sufficient amounts of oil, but the oil has low levels of the specific desired fatty acids. Nevertheless, the field of oil modification in plants is wide and a number of different products have already been designed. Rape oil containing lauric acid has been marketed, and soybeans with modified levels of unsaturated fatty acids are available. In some cases the production of speciality oils seems to be straight-forward. In others, however, a number of unexpected complications have arisen which have hampered the production of plants capable of making some specific oils. For example, mutations in plant lipid synthesis genes are generally difficult to detect due to the pleiotrophic effects of mutations on plant hardiness and yield. Even if detected, proteins involved in pathways of interest have proved difficult to isolate due to their biochemical instability. Where regulation of such proteins has been successfully altered, results generally do not coincide with expectations, presumably due to the effect of multiple converging pathways. Examples of such problems relating to the production of Arabidopsis producing petroselinic acid are disclosed in Ohlrogge, 13th International Symposium on Plant Lipids, Seville, Spain: 219 & 801, (1998). Thus, there is considerable work yet to be done in achieving reliable, large-scale production of a range of commercially desirable oils.
Broadly speaking, there are two main approaches to altering the lipid content of an oil, which to date have been applied as alternatives. Firstly, plants may be modified to produce fatty acids which are foreign to the native plant. For example, rape may be modified to produce laureate which is not naturally produced by that plant. Secondly, the pattern and/or extent of incorporation of fatty acids into the glycerol backbone of a lipid may be altered.
Lipids are formed by the addition of the fatty acid moieties into the glycerol backbone by acyltransferase enzymes. There are three positions on the glycerol backbone at which fatty acids may be introduced. The acyltransferase enzymes which are specific for each position are hence referred to as 1-, 2-, and 3-acyltransferase enzymes respectively.
One of the aims of lipid engineering is to produce oils which are high in erucic (22: 1) acid. Such oils are desirable for a number of reasons, in particular as replacements and/or substitutes for mineral oils, as described above. In the case of Brassica napus one of the most commercially important crops cultivated today, and other oil seed Brassica species, e.g. Brassica juncea, the 2-acyltransferase positively discriminates against the incorporation of erucic acid in the second position. Thus, even in those crops where erucic acid is incorporated into the first and third positions, only a maximum of 66% of the fatty acids of the lipid can be erucic acid. These latter varieties of rape are nevertheless known as HEAR (high erucic acid rape) varieties.
It is desirable to further increase the erucic acid content of both HEAR varieties, and other useful vegetable oil crops, for example maize, sunflower, soya, mustards and linseed. Genes encoding 2-acyltransferases have been introduced into plants, in order to try to incorporate erucic acid into the second position of the glycerol backbone, with the aim of increasing the overall erucic acid content in a lipid (Brough et al., Mol Breeding 2: 133-142 (1996)). This was successful in the re-distribution of erucic acid in the triglyceride but has not increased the overall erucic acid content of the oil. One possible reason for this is that the levels of “free” erucic acid available for incorporation into lipids in a plant are too low to support high levels of trierucin synthesis (Millar et al., The Plant J. 12(1) 121-131 (1997)). Thus, knowledge of the factors involved in the regulation of erucic acid levels in a plant is being sought.
In this text, the terms “free” or “available” erucic acid mean erucic acid which has not been incorporated into lipid. References to the erucic acid content of oil means that which has been incorporated into lipid.
Biochemical and genetic studies have elucidated most of the pathways involved in the production of vegetable oils (Ohlrogge & Browse, Plant Cell 7: 957-970, 1995). An enzyme involved in the synthesis of fatty acids is the fatty acid elongation enzyme (FAE) complex, also referred to as an elongase. This enzyme complex is responsible for the conversion of fatty acids 18 carbons long, such as oleic (18:1) acid, to fatty acids known as very long chain fatty acids, which include erucic acid (22: 1). Given its involvement in the production of erucic acid, it is apparent that the elongase plays a role in regulation of the levels of free erucic acid in a plant. Thus, it has been suggested that, in relation to Arabidopsis, over-expression of the FAE1 gene may assist in obtaining higher levels of free erucic acid (Millar et al., The Plant J. 12(1) 121-131 (1997)). Depending upon the plant species, the products of this enzyme are C20 and C22 saturated fatty acids, utilised in wax production in leek, or C20 to C24 monounsaturated fatty acids utilised as seed storage oils in crucifers.
Over recent years, a number of β-keto-acyl-CoA synthetase (“elongase”) genes, in addition to the Arabidopsis FAE1 gene, have been cloned from a variety of species. Sequence data is available for elongases isolated from Arabidopsis (Millar & Kunst, Plant J. 12: 121-131, (1997)), jojoba (Lassner et al, Plant Cell 8: 281-292, (1996)), honesty (Millar & Kunst, Plant J. 12: 121-131, (1997)), leek (Evenson & Post-Beittenmiller, Plant Physiol. 109: 707-716, (1995)) and oilseed rape (Sequence: Genbank AF009563 & BNU50771). These enzymes all produce a range of other very long chain fatty acids besides erucic acid, for example fatty acid 20:1 in Arabidopsis and oilseed rape, fatty acids 20:0 and 22:0 in leek and fatty acid 24:1 in honesty and jojoba.
Recently, experiments have been performed on high-erucic acid rapeseed plants in which the plants were transformed with constructs encoding an acyltransferase and an elongase. However, none of the transformants were found to contain erucic acid levels greater than 60% (Han et al., Plant Mol. Bio. 46 229-239 (2001)).